Confocal Microscopy List

Re: aliquoting 4% formaldehyde

Posted by Sylvie Le Guyader on
URL: http://confocal-microscopy-list.275.s1.nabble.com/aliquoting-4-formaldehyde-tp7585663p7585675.html

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

From my experience of working with filopodia which retract/detach very easily:

Prepare 8% formaldehyde in PBS from powder (should be completely dissolved) and freeze it at -20 deg C in 15ml tubes (low volume/surface ratio). This is stable litterally for years. When needed, thaw an aliquot and keep it in the fridge for about 2 months. Warm up just the amount you need to 37 deg C, add it Vol/vol to your warm sample without shocking the cells by rinsing them with PBS. Incubate 5 min at 37 deg C (for a cell monolayer). then rinse with PBS.

It is easy to spot if cells in a monolayer suffer during fixation. Filopodia are not straight, cells detach easily during gentle washing, parts of the cell fold onto the cells. See an example here.

lately some of our users started using small capsules of pre made 4% PFA. Apart from the terrible waste in packaging, we have encountered a much higher than normal number of cases of folded cells.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Hälsovägen 7,

Novum, G lift, floor 6

14157 Huddinge

Sweden

mobile: +46 (0) 73 733 5008

office: +46 (0) 08-524 811 72 New number!

LCI website

From: Confocal Microscopy List [[hidden email]] on behalf of Cromey, Douglas W - (dcromey) [[hidden email]]
Sent: 20 September 2016 16:34
To: [hidden email]
Subject: aliquoting 4% formaldehyde

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I was talking with a student yesterday about their cells which did not look particularly good under the microscope. The discussion eventually came around to fixation. The student told me that he made up some 4% formaldehyde fixative and then froze back (20 deg C) aliquots for later use. This was something he had seem on an online research forum. In my fairly lengthy experience I have never heard of this being done and I am suspicious that this is NOT a good idea. Thoughts?

Doug

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA

office: LSN 463 email: [hidden email]

voice: 520-626-2824 fax: 520-626-2097

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

UA Microscopy Alliance - http://microscopy.arizona.edu